RESUMO
In contrast to peas (Pisum sativum), where mitochondrial lipoamide dehydrogenase is encoded by a single gene and shared between the alpha-ketoacid dehydrogenase complexes and the Gly decarboxylase complex, Arabidopsis has two genes encoding for two mitochondrial lipoamide dehydrogenases. Northern-blot analysis revealed different levels of RNA expression for the two genes in different organs; mtLPD1 had higher RNA levels in green leaves compared with the much lower level in roots. The mRNA for mtLPD2 shows the inverse pattern. The other organs examined showed nearly equal RNA expressions for both genes. Analysis of etiolated seedlings transferred to light showed a strong induction of RNA expression for mtLPD1 but only a moderate induction of mtLPD2. Based on the organ and light-dependent expression patterns, we hypothesize that mtLPD1 encodes the protein most often associated with the Gly decarboxylase complex, and mtLPD2 encodes the protein incorporated into alpha-ketoacid dehydrogenase complexes. Due to the high level of sequence conservation between the two mtLPDs, we assume that the proteins, once in the mitochondrial matrix, are interchangeable among the different multienzyme complexes. If present at high levels, one mtLPD might substitute for the other. Supporting this hypothesis are results obtained with a T-DNA knockout mutant, mtlpd2, which shows no apparent phenotypic change under laboratory growth conditions. This indicates that mtLPD1 can substitute for mtLPD2 and associate with all these multienzyme complexes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Di-Hidrolipoamida Desidrogenase/genética , Mitocôndrias/genética , Complexos Multienzimáticos/genética , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA Complementar , DNA Mitocondrial , DNA de Plantas , Di-Hidrolipoamida Desidrogenase/metabolismo , Regulação da Expressão Gênica de Plantas , Glicina Desidrogenase (Descarboxilante) , Mitocôndrias/enzimologia , Complexos Multienzimáticos/metabolismo , Pisum sativum/enzimologia , Pisum sativum/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/metabolismo , Transdução de SinaisRESUMO
Lipoamide dehydrogenase is a subunit of the alpha-ketoacid dehydrogenases and the glycine decarboxylase complex in mitochondria, and the pyruvate dehydrogenase complex in plastids. We report here the unexpected finding of two plastidic isoforms of lipoamide dehydrogenase from Arabidopsis thaliana that are different from the mitochondrial form of the enzyme. The cDNA clones were confirmed by sequence alignment analysis and their location verified by chloroplast import assay. They are single copy genes that appear to be expressed in parallel in different tissues with highest level in developing siliques. Phylogenetic analysis gives further exemplary evidence for the plastidic evolution derived from cyanobacteria.
Assuntos
Arabidopsis/enzimologia , Di-Hidrolipoamida Desidrogenase/metabolismo , Plastídeos/enzimologia , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Núcleo Celular/genética , Cloroplastos/enzimologia , Cloroplastos/genética , Cloroplastos/metabolismo , Clonagem Molecular , Cianobactérias/enzimologia , Di-Hidrolipoamida Desidrogenase/química , Di-Hidrolipoamida Desidrogenase/genética , Genes de Plantas/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Pisum sativum/citologia , Pisum sativum/metabolismo , Filogenia , Plastídeos/genética , Plastídeos/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA de Plantas/análise , RNA de Plantas/genética , Alinhamento de SequênciaRESUMO
A streamlined mini binary vector was constructed that is less than 1/2 the size of the pBIN19 backbone (3.5 kb). This was accomplished by eliminating over 5 kb of non-T-DNA sequences from the pBIN19 vector. The vector still retains all the essential elements required for a binary vector. These include a RK2 replication origin, the nptIII gene conferring kanamycin resistance in bacteria, both the right and left T-DNA borders, and a multiple cloning site (MCS) in between the T-DNA borders to facilitate cloning. Due to the reduced size, more unique restriction sites are available in the MCS, thus allowing more versatile cloning. Since the traF region was not included, it is not possible to mobilize this binary vector into Agrobacterium by triparental mating. This problem can be easily resolved by direct transformation. The mini binary vector has been demonstrated to successfully transform Arabidopsis plants. Based on this mini binary vector, a series of binary vectors were constructed for plant transformation.
Assuntos
Vetores Genéticos/genética , Plantas/genética , Transformação Genética , Agrobacterium tumefaciens/genética , Arabidopsis/genética , Southern Blotting , Clonagem Molecular , DNA Bacteriano/genética , DNA de Plantas/genética , Vetores Genéticos/química , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Experiments in Antirrinum majus, Arabidopsis thaliana and Petunia hybrida have demonstrated that putative transcription factors of the so-called MADS-box family play an important role in determining floral organ identity. Such regulatory genes are transiently expressed in small numbers of cells in the floral apex. Here we describe the isolation of a cDNA from Nicotiana tabacum coding for a MADS-box protein which is expressed in both the floral and vegetative organs of the plant.
